Lasers are an ideal source to illuminate cellular samples for analysis due to their high brightness, monochromaticity and coherence. Although there are different spatial modes for laser light, the most common is the TEM00 mode in which laser light is generated with a Gaussian intensity profile. The Gaussian intensity profile is governed by the equation I=Io*e−2(r/w)2, where “Io” is the peak intensity point, “w” is the waist radius (the point where the intensity is 13.5% of the peak intensity) and “r” is distance from the peak. Per the equation, the intensity of the beam begins to fall off immediately on either side of the peak. Assuming a beam waist 85 microns in diameter, the intensity drops to 97% of the peak value at a point just 5.0 microns from the center of the beam. At a point 10 microns from center, the intensity drops to 89% of the peak value. Therefore, when illuminating a core stream containing cells in motion, as is done in flow cytometry, a cell disposed just 5 to 10 microns from the center of the core stream will see a significantly lower level of irradiation and therefore, generate less fluorescence. Because the location of cells in the core stream can vary by these amounts in normal operation of a typical flow cytometer, the laser intensity profile across the beam is a source of measurement variation in these instruments.
In flow cytometry, the laser beam diameter is maintained large relative to the cell and core size, in order to minimize cell-to-cell fluorescence intensity measurement variation. The typical beam size is about 85 microns in diameter for a theoretical core stream size of 10 microns. If the core stream is made larger, then the beam must also be made larger to maintain illumination uniformity. It should be noted that making the core larger may lead to a much higher amount of coincident events in a standard flow cytometer. Although coincidence is not an issue in an imaging flow cytometer, an increase in core size may lead to defocus, which may degrade the imagery. Although making the beam approximately ten times larger than the cell decreases measurement variation, it also wastes a considerable amount of light, thereby decreasing the sensitivity of the instrument, unless this decrease is offset with a higher power and concomitantly higher cost laser. It is therefore desirable to use a higher proportion of the laser light to illuminate a core stream in a flow cytometer without incurring an increase in measurement variation, which should decrease instrument cost by enabling the use of a lower-power laser, or alternatively, increase sensitivity if the same power laser is used. It would also be desirable to actively enable tailoring of the beam profile to further increase sensitivity, where higher amounts of variation are acceptable.
There have been many attempts over the past two decades to generate what are commonly called “flat top” laser profiles to reduce intensity variation near the center of the laser beam. One elegant approach uses diffractive optics, which can be designed to generate a wide array of profiles, including a flat top. Although this approach is effective at generating a flat top, it suffers from the drawback of light loss, reducing overall intensity, and therefore, negating much of the benefit relative to simply increasing beam size.
Another approach disclosed in U.S. Pat. No. 4,826,299 uses a double wedge-shaped optic that is disposed in the laser beam path. The laser beam is then significantly expanded before being imaged into the flow cell. The net effect is the generation of a nearly flat top intensity profile. This method does not suffer the intensity loss associated with diffractive optics and therefore generates a higher intensity than the Gaussian intensity profile with a flat top over the region of interest. When properly designed, this technique can generate a flat top which is 1.5 to 2 times more intense than the standard Gaussian intensity profile, over a 10 micron region around the center of the beam. However, this technique also has several drawbacks. First, the method uses highly specialized optics and requires a substantial expansion of the beam prior to imaging into the flow cell, which adds cost and poses additional constraints on the optical and mechanical design of the instrument. While these issues may theoretically be overcome at a lower cost than using a higher powered laser, this technique relies on superimposing one part of the beam with another in order to smooth out the profile. Since laser light has a high degree of coherence, in practice, the overlapping beams can constructively and/or destructively interfere with each other, generating significant perturbations in the profile. Once the beam is aligned, and a uniform profile is established, very small changes in the position of the beam on the final imaging lens may lead to significant perturbations in the intensity profile. Therefore, very small thermal changes or pointing errors can lead to a loss of uniformity and negate the benefits of the method. Accordingly, an alternative approach is needed to produce a laser beam having a flat top Gaussian beam intensity profile that avoids these problems, and thus, to achieve the benefits of using such a laser beam, as noted above.